The acrosome of a sperm must undergo exocytosis before the sperm can fertilize an egg. Ejaculated mammalian sperm become capable of exocytosis during a poorly understood maturation process. The long-term objective of this project is to understand how acrosomal exocytosis is controlled. Sperm of some infertile men are unable to have normal acrosomal exocytosis, and a full understanding of how acrosomal exocytosis is controlled may suggest new ways to control fertility. Sperm lipids play an important role in the control of sperm exocytosis. Sperm must lose cholesterol to become acrosomally responsive. Loss of cholesterol increases the intracellular pH. Experiments will determine how human sperm pH is controlled so the mechanism of cholesterol's effect can be better understood. A structure-activity analysis will be performed to reveal what features of the cholesterol molecule are required for activity. The importance of cholesterol's ability to promote order among phospholipids will be studied by measuring fluorescence anisotropy of a membrane probe. A second lipid, sphingomyelin, has recently been shown to be involved in the control of acrosomal exocytosis, and experiments will test whether it acts by controlling cholesterol efflux or by serving as the source of signalling molecules. The cholesterol content of freshly ejaculated and oviductal bovine sperm will be compared to see if sperm lose cholesterol in the female tract.